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BMP Inhibition in Seminomas Initiates Acquisition of Pluripotency via NODAL Signaling Resulting in Reprogramming to an Embryonal Carcinoma.

Nettersheim D, Jostes S, Sharma R, Schneider S, Hofmann A, Ferreira HJ, Hoffmann P, Kristiansen G, Esteller MB, Schorle H - PLoS Genet. (2015)

Bottom Line: Analysis of expression and DNA methylation dynamics during transition of TCam-2 revealed that many pluripotency- and reprogramming-associated genes were upregulated while seminoma-markers were downregulated.We demonstrate that inhibition of BMP signaling is the initial event in reprogramming, resulting in activation of the pluripotency-associated genes and NODAL signaling.In parallel, DNMT3B-driven de novo methylation silences seminoma-associated genes and epigenetically fixes the EC state.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pathology, Department of Developmental Pathology, University Medical School, Bonn, Germany.

ABSTRACT
Type II germ cell cancers (GCC) can be subdivided into seminomas and non-seminomas. Seminomas are similar to carcinoma in situ (CIS) cells, the common precursor of type II GCCs, with regard to epigenetics and expression, while embryonal carcinomas (EC) are totipotent and differentiate into teratomas, yolk-sac tumors and choriocarcinomas. GCCs can present as seminomas with a non-seminoma component, raising the question if a CIS gives rise to seminomas and ECs at the same time or whether seminomas can be reprogrammed to ECs. In this study, we utilized the seminoma cell line TCam-2 that acquires an EC-like status after xenografting into the murine flank as a model for a seminoma to EC transition and screened for factors initiating and driving this process. Analysis of expression and DNA methylation dynamics during transition of TCam-2 revealed that many pluripotency- and reprogramming-associated genes were upregulated while seminoma-markers were downregulated. Changes in expression level of 53 genes inversely correlated to changes in DNA methylation. Interestingly, after xenotransplantation 6 genes (GDF3, NODAL, DNMT3B, DPPA3, GAL, AK3L1) were rapidly induced, followed by demethylation of their genomic loci, suggesting that these 6 genes are poised for expression driving the reprogramming. We demonstrate that inhibition of BMP signaling is the initial event in reprogramming, resulting in activation of the pluripotency-associated genes and NODAL signaling. We propose that reprogramming of seminomas to ECs is a multi-step process. Initially, the microenvironment causes inhibition of BMP signaling, leading to induction of NODAL signaling. During a maturation phase, a fast acting NODAL loop stimulates its own activity and temporarily inhibits BMP signaling. During the stabilization phase, a slow acting NODAL loop, involving WNTs re-establishes BMP signaling and the pluripotency circuitry. In parallel, DNMT3B-driven de novo methylation silences seminoma-associated genes and epigenetically fixes the EC state.

No MeSH data available.


Related in: MedlinePlus

Detailed analyses of Gex and 5mC dynamics during reprogramming of TCam-2 cells.(A) Histogram showing changes in Gex 1–6 weeks after xenografting compared to TCam-2 in vitro. (B) Violin plot illustrating 5mC level distribution of all differentially methylated genes during SET. (C) Circos diagrams illustrate inverse correlation between Gex and 5mC during SET and in comparison to 2102EP cells in the 53 5mC /Gex-group genes. Gex and 5mC data of each analyzed gene is linked to each analyzed sample—the thicker a connection the higher the Gex /5mC level and vice versa. Thus, genes with high 5mC levels in a certain sample (thick connection) show a small connection in the illustration of Gex data. T i.v. = TCam-2 in vitro, 1w = TCam-2 in vivo 1w, 6w = TCam-2 in vivo 6w, 2102EP = 2102EP in vitro. (D) Volcano plot of Gex and 5mC data of the 5mC /Gex-group 1 and 6 weeks after xenografting. (E) Gex and 5mC dynamis of indicated genes during SET. Red framed black squares = Gex, blue framed black squares = 5mC of averaged data of 2102EP in vivo 4 /8 weeks.
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pgen.1005415.g002: Detailed analyses of Gex and 5mC dynamics during reprogramming of TCam-2 cells.(A) Histogram showing changes in Gex 1–6 weeks after xenografting compared to TCam-2 in vitro. (B) Violin plot illustrating 5mC level distribution of all differentially methylated genes during SET. (C) Circos diagrams illustrate inverse correlation between Gex and 5mC during SET and in comparison to 2102EP cells in the 53 5mC /Gex-group genes. Gex and 5mC data of each analyzed gene is linked to each analyzed sample—the thicker a connection the higher the Gex /5mC level and vice versa. Thus, genes with high 5mC levels in a certain sample (thick connection) show a small connection in the illustration of Gex data. T i.v. = TCam-2 in vitro, 1w = TCam-2 in vivo 1w, 6w = TCam-2 in vivo 6w, 2102EP = 2102EP in vitro. (D) Volcano plot of Gex and 5mC data of the 5mC /Gex-group 1 and 6 weeks after xenografting. (E) Gex and 5mC dynamis of indicated genes during SET. Red framed black squares = Gex, blue framed black squares = 5mC of averaged data of 2102EP in vivo 4 /8 weeks.

Mentions: Next, we distinguished CpG-island-associated DNA methylation events from DNA methylation at open sea context (i. e. non-CpG-island context) (Fig 2A and 2B). In TCam-2, the vast majority of CpG-island-associated CpGs show low levels of DNA methylation in regions 1500 and 200 bp upstream of the TSS, the 5’-UTR and the 1st exon (orange circle in S2A Fig), while CpG-islands within the gene body and the 3’-UTR appear medium to hypermethylated (green circle in S2A Fig). Six weeks after xenografting the CpGs in the gene body display distinct changes in methylation (red circle in S2A Fig) and demethylation (yellow circle in S2A Fig), while probes within the TSS200 /1500, 5’-UTR and 1st exon remain hypomethylated or become demethylated (black circle in S2A Fig).


BMP Inhibition in Seminomas Initiates Acquisition of Pluripotency via NODAL Signaling Resulting in Reprogramming to an Embryonal Carcinoma.

Nettersheim D, Jostes S, Sharma R, Schneider S, Hofmann A, Ferreira HJ, Hoffmann P, Kristiansen G, Esteller MB, Schorle H - PLoS Genet. (2015)

Detailed analyses of Gex and 5mC dynamics during reprogramming of TCam-2 cells.(A) Histogram showing changes in Gex 1–6 weeks after xenografting compared to TCam-2 in vitro. (B) Violin plot illustrating 5mC level distribution of all differentially methylated genes during SET. (C) Circos diagrams illustrate inverse correlation between Gex and 5mC during SET and in comparison to 2102EP cells in the 53 5mC /Gex-group genes. Gex and 5mC data of each analyzed gene is linked to each analyzed sample—the thicker a connection the higher the Gex /5mC level and vice versa. Thus, genes with high 5mC levels in a certain sample (thick connection) show a small connection in the illustration of Gex data. T i.v. = TCam-2 in vitro, 1w = TCam-2 in vivo 1w, 6w = TCam-2 in vivo 6w, 2102EP = 2102EP in vitro. (D) Volcano plot of Gex and 5mC data of the 5mC /Gex-group 1 and 6 weeks after xenografting. (E) Gex and 5mC dynamis of indicated genes during SET. Red framed black squares = Gex, blue framed black squares = 5mC of averaged data of 2102EP in vivo 4 /8 weeks.
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Related In: Results  -  Collection

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pgen.1005415.g002: Detailed analyses of Gex and 5mC dynamics during reprogramming of TCam-2 cells.(A) Histogram showing changes in Gex 1–6 weeks after xenografting compared to TCam-2 in vitro. (B) Violin plot illustrating 5mC level distribution of all differentially methylated genes during SET. (C) Circos diagrams illustrate inverse correlation between Gex and 5mC during SET and in comparison to 2102EP cells in the 53 5mC /Gex-group genes. Gex and 5mC data of each analyzed gene is linked to each analyzed sample—the thicker a connection the higher the Gex /5mC level and vice versa. Thus, genes with high 5mC levels in a certain sample (thick connection) show a small connection in the illustration of Gex data. T i.v. = TCam-2 in vitro, 1w = TCam-2 in vivo 1w, 6w = TCam-2 in vivo 6w, 2102EP = 2102EP in vitro. (D) Volcano plot of Gex and 5mC data of the 5mC /Gex-group 1 and 6 weeks after xenografting. (E) Gex and 5mC dynamis of indicated genes during SET. Red framed black squares = Gex, blue framed black squares = 5mC of averaged data of 2102EP in vivo 4 /8 weeks.
Mentions: Next, we distinguished CpG-island-associated DNA methylation events from DNA methylation at open sea context (i. e. non-CpG-island context) (Fig 2A and 2B). In TCam-2, the vast majority of CpG-island-associated CpGs show low levels of DNA methylation in regions 1500 and 200 bp upstream of the TSS, the 5’-UTR and the 1st exon (orange circle in S2A Fig), while CpG-islands within the gene body and the 3’-UTR appear medium to hypermethylated (green circle in S2A Fig). Six weeks after xenografting the CpGs in the gene body display distinct changes in methylation (red circle in S2A Fig) and demethylation (yellow circle in S2A Fig), while probes within the TSS200 /1500, 5’-UTR and 1st exon remain hypomethylated or become demethylated (black circle in S2A Fig).

Bottom Line: Analysis of expression and DNA methylation dynamics during transition of TCam-2 revealed that many pluripotency- and reprogramming-associated genes were upregulated while seminoma-markers were downregulated.We demonstrate that inhibition of BMP signaling is the initial event in reprogramming, resulting in activation of the pluripotency-associated genes and NODAL signaling.In parallel, DNMT3B-driven de novo methylation silences seminoma-associated genes and epigenetically fixes the EC state.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pathology, Department of Developmental Pathology, University Medical School, Bonn, Germany.

ABSTRACT
Type II germ cell cancers (GCC) can be subdivided into seminomas and non-seminomas. Seminomas are similar to carcinoma in situ (CIS) cells, the common precursor of type II GCCs, with regard to epigenetics and expression, while embryonal carcinomas (EC) are totipotent and differentiate into teratomas, yolk-sac tumors and choriocarcinomas. GCCs can present as seminomas with a non-seminoma component, raising the question if a CIS gives rise to seminomas and ECs at the same time or whether seminomas can be reprogrammed to ECs. In this study, we utilized the seminoma cell line TCam-2 that acquires an EC-like status after xenografting into the murine flank as a model for a seminoma to EC transition and screened for factors initiating and driving this process. Analysis of expression and DNA methylation dynamics during transition of TCam-2 revealed that many pluripotency- and reprogramming-associated genes were upregulated while seminoma-markers were downregulated. Changes in expression level of 53 genes inversely correlated to changes in DNA methylation. Interestingly, after xenotransplantation 6 genes (GDF3, NODAL, DNMT3B, DPPA3, GAL, AK3L1) were rapidly induced, followed by demethylation of their genomic loci, suggesting that these 6 genes are poised for expression driving the reprogramming. We demonstrate that inhibition of BMP signaling is the initial event in reprogramming, resulting in activation of the pluripotency-associated genes and NODAL signaling. We propose that reprogramming of seminomas to ECs is a multi-step process. Initially, the microenvironment causes inhibition of BMP signaling, leading to induction of NODAL signaling. During a maturation phase, a fast acting NODAL loop stimulates its own activity and temporarily inhibits BMP signaling. During the stabilization phase, a slow acting NODAL loop, involving WNTs re-establishes BMP signaling and the pluripotency circuitry. In parallel, DNMT3B-driven de novo methylation silences seminoma-associated genes and epigenetically fixes the EC state.

No MeSH data available.


Related in: MedlinePlus